1. Field of the Invention
This invention relates to determination of the concentration of a ligand in a sample, more particularly, a ligand that specifically binds to a receptor.
2. Discussion of the Art
For the past several decades, immunoassays have been performed using fluorescence, chemiluminescence, or other means of generating a signal in response to an analyte. Currently, many immunoassays are performed by measurement of the intensity of a light signal generated in the total volume of a reaction mixture. The light signal generated can be measured by an optical means, wherein the light signal generated is emitted by a large number of molecules. In a typical embodiment, these immunoassays can be carried out by combining a sample suspected of containing an antigen with a reagent comprising a first antibody attached to a solid support, e.g., a bead, a microparticle, to form a reaction mixture. The antigen, if present in the sample, specifically binds to the first antibody. A conjugate, which comprises a second antibody having a label attached thereto, is introduced to the reaction mixture and specifically binds to the antigen, which is specifically bound to the first antibody, which, as stated previously, is attached to the solid support. Such an immunoassay is referred to as a sandwich immunoassay or an immunometric assay. This type of immunoassay is shown schematically in FIG. 1. The signal attributable to the label is then measured after unbound conjugate is removed from the reaction mixture, typically by performing a wash step. The signal that is derived from the total volume of the reaction mixture is measured and then compared to a calibration curve to establish the concentration of antigen present in the sample.
Another type of immunoassay is called a competitive immunoassay. In a typical embodiment, an unlabeled antigen and a labeled antigen compete for the same antibody site. Alternatively, an antibody and a labeled antibody compete for the same antigen site. In an example of the former, a labeled antigen and an unlabeled antigen are used. A solid support is coated with an antibody that can specifically bind to either the labeled antigen or to the unlabeled antigen. The solid support, the labeled antigen, and a patient's sample suspected of containing the antigen are combined. Of course, any antigen in the patient's sample is unlabeled. The labeled antigen and the unlabeled antigen compete for antibody sites on the solid support. Only when the labeled antigen attaches to the antibody on the solid support can a signal be produced, because only the labeled antigen can generate a signal. The amount of antigen in the patient's sample is inversely proportional to the amount of signal produced. This type of immunoassay is shown schematically in FIG. 2.
In performing immunoassays using different optical methods, a number of parameters must be considered. These parameters include the time required to perform the immunoassay, the amount of sample needed to carry out the immunoassay, the amount of additional reagents needed to carry out the immunoassay, the number of steps needed to complete the immunoassay, the sensitivity of the immunoassay, and the dynamic range of the immunoassay. The dynamic range can often cover three or more orders of magnitude. For many decades, immunoassays that utilize magnetic microparticles have been shown to provide adequate values for most of the parameters mentioned previously. Magnetic microparticles allow separation of analyte bound to conjugate from unbound conjugate and other reagents in a simple manner. Another attractive property of magnetic microparticles is that they can easily be controlled in a solution in order to allow for binding of analyte or conjugate in the solid phase in a relatively short time. By making use of magnetic attraction, magnetic microparticles can be moved and washed in order to provide information about only the analyte bound to the magnetic microparticles.
A major drawback with the use of any microparticle as the solid support is lack of uniformity from microparticle to microparticle with respect to the amount of antibody coated on the microparticle. Another drawback is the undesired interaction of the conjugate with the microparticles. Such undesired interaction may affect results of the immunoassay and, consequently, may require extensive study of a number of different microparticles, made by different manufacturers, for use on an immunoassay analyzer. Another drawback presents itself when immunoassays are performed in a reaction vessel. The conjugate can bind to the surfaces of the reaction vessel, which is undesirable. These drawbacks not only limit sensitivity of an immunoassay, but can yield false results upon measurement of the analyte.
Additional problems that may arise in immunoassays involve (a) non-specific binding of the conjugate to the solid support and (b) aggregation of reagents. These problems are a major concern to developers of an assay. Typically, methods to reduce non-specific binding involve not only tailoring of reagents, but also mixing them in appropriate proportions to provide the desired results. These methods, which entail a significant amount of trial and error, often result in making development of an assay a long process, as well as making development of an assay an empirical process, with the result that reagents often vary from one lot to another lot. Moreover, a signal resulting from non-specific binding of the conjugate can be higher than the signal resulting from specific binding of the conjugate to an analyte, thereby limiting the sensitivity of the immunoassay. Only through the use of calibration using samples free of any analyte can the effects of non-specific binding during an actual assay be estimated.
Another potential drawback of a typical immunoassay is that after the assay is performed, the only record of the assay is the value of signal. There is no opportunity to recheck the sample for defects or to obtain more information if new methods of analysis become available. Not only will there be no record of the properties of the solid phase, there will also be no way to review recorded data using a newly developed algorithm. The ability to use historical data in order to extract new information is not possible.
Therefore, a need exists to develop analytical instruments and methods for addressing non-specific binding and undesired performance of the solid phase in a given assay while simultaneously acquiring data from the assay to improve sensitivity of the assay. A need exists to reduce use of reagents and provide measurement in an adequate time for use in both a laboratory setting and a point-of-care setting. In principle, such a method would also provide real-time quality control as the assay is being performed. Furthermore, it is desired to alleviate the need to generate new calibration curves and to reduce the variation of reagents from lot to lot. It would also be desirable to maintain a record of the assay in such a manner that it can be reviewed at a later date through the use of different methods as these methods become available.
New detection methods can be coupled with devices from the emerging fields of nanotechnology and microfluidics to provide smaller, more effective, and more sensitive assays for detecting and measuring analytes in biological samples.